Chemical composition and mechanical properties of plant materials determine to a major extent how those plant materials are utilized. Cell wall content and composition account for most of the variation in mechanical strength of plant tissues. Also, cell wall composition is a major determinant of silage quality. Cell walls constitute a major sink in the vegetative parts of plants, accounting, for example, for approximately 80% of the corn stalk (FIG. 1). For the whole corn plant, including grain, cell wall accounts for approximately 35–40% of the dry mass.
Cellulose, the most abundant organic molecule on Earth, is made at the plasma membrane and directly deposited into the cell wall [Ray, et al., (1976), Ber. Deutsch. Bot. Ges. Bd. 89:121–146]. By inter- and intra-chain hydrogen bonding, β-1,4-glucan chains form para-crystalline microfibrils which eventually form ribbons and fibers, giving cellulose a very high tensile strength [Niklas (1992), “Plant biomechanics: An engineering approach to plant form and function,” The University of Chicago Press, p. 607]. Because of its para-crystalline nature, cellulose makes a disproportionately greater contribution toward tensile strength of plant tissues than it would if it were amorphous in nature.
Cell wall of a maize stalk consists mostly of cellulose and hemicellulose, with lignin constituting a minor proportion, i.e., ˜10% (FIG. 1). In a study conducted on three contrasting pairs of hybrids, we have determined that cellulose concentration in a unit length of stalk below the ear is correlated with tensile strength of the stalk (FIG. 2). Stalk lodging is a major problem in maize, accounting for significant yield losses. Increasing cellulose concentration in the wall will result in a mechanically stronger tissue, reducing the problem of stalk lodging.
The rate of cellulose synthesis exerts major control on the formation of the rest of the wall, as cellulose is its dominant constituent (FIG. 1). Formation of UDP-glucose, the substrate for cellulose synthase (CesA), occurs through two pathways in plants: one through UDP-glucose pyrophosphorylase (UGPase) and the other through sucrose synthase (FIG. 3). Sucrose synthase (SuSy) catalyzes the reversible reaction:                Sucrose+UDP⇄UDP−Glucose+FructoseThus, the cleavage reaction provides the precursor for synthesis of starch and cellulose. SuSy uses the energy of the glycosidic bond from sucrose to make UDP-glucose from UDP, releasing fructose in the process; fructose can then be channeled into UDP-glucose by the UGPase pathway (FIG. 3). While sucrose synthase has historically been considered active in the cytoplasm of plant cells, Amor et al. found tight association of about half of the total cellular SuSy with the plasma membrane in cotton and suggested that SuSy might channel substrate directly from sucrose to CesA [Amor, et al., (1995), “A membrane-associated form of sucrose synthase and its potential role in synthesis of cellulose and callose in plants,” Proc. Natl. Acad. Sci. USA 92:9353–9357]. Therefore, in a sink tissue, such as growing corn stalk, sucrose synthase provides an economical route for the formation of UDP-glucose from sucrose. In contrast, the UGPase pathway utilizes more energy in the form of nucleotide triphosphates to produce UDP-glucose from hexose sugars.        
Until the present invention, only two sucrose synthase genes have been known in maize, shrunken-1 (Sh1) and constitutive sucrose synthase (Sus1), both of which map to chromosome 9 [Huang, et al. (1994), “Complete nucleotide sequence of the maize (Zea mays L.) sucrose synthase 2 cDNA,” Plant Physiology Rockville 104:293–294; McCarty, et al. (1986), “The cloning, genetic mapping and expression of the constitutive sucrose synthase locus of maize,” Proc. Natl. Acad. Sci. USA 83:9099–9103; Werr, et al. (1985), “Structure of the sucrose synthase (EC 2.4.1.13) gene on chromosome 9 of Zea mays,” EMBO J. 4:1373–1380]. These paralogs encode the sucrose synthase isozymes SS1 and SS2, respectively.
Membrane-associated SuSy has also been found in carrot and maize [Carlson, et al. (1996), “Evidence for plasma membrane-associated forms of sucrose synthase in maize,” Molecular and General Genetics 252:303–310; Sturm, et al. (1999), “Tissue-specific expression of two genes for sucrose synthase in carrot (daucus carota L.),” Plant Molecular Biology 39:349–360]. Both the known forms of SuSy in maize were found to be associated with the plasma membrane fraction from developing endosperm. Interestingly, Sh1 was suggested to play a greater role in cell wall formation than the constitutive sucrose synthase (Sus1), which was purported to contribute more toward starch formation [Chourey, et al. (1998), “Genetic evidence that the two isozymes of sucrose synthase present in developing maize endosperm are critical, one for cell integrity and the other for starch biosyntheses,” Molecular and General Genetics 259:88–96]. SuSy is known to become reversibly phosphorylated at a unique seryl residue [Huber, et al. (1996), “Phosphorylation of serine-15 of maize leaf sucrose synthase,” Plant Physiology Rockville 112:793–802]. The unphosphorylated form, because of its relatively greater surface hydrophobicity, is favored to bind the membrane [Winter, et al. (1997), “Membrane association of sucrose synthase: Changes during the graviresponse and possible control by protein phosphorylation,” FEBS Letters 420:151–155].
Sucrose synthase has been suggested to channel substrate to the matrix polysaccharide synthases, based on association with Golgi and a previous report of its involvement in cellulose synthesis [Buckeridge, et al., (1999), “The mechanism of synthesis of a mixed-linkage (1fwdarw3), (1fwdarw4) beta-D-glucan in maize. Evidence for multiple sites of glucosyl transfer in the synthase complex,” Plant-Physiology-Rockville 120:1105–1116 ]. Direct evidence for the contribution of SuSy toward substrate generation for cellulose synthesis was provided by Nakai et al [Nakai, et al. (1999), “Enhancement of cellulose production by expression of sucrose synthase in Acetobacter xylinum,” Proc. Natl. Acad. Sci. USA 96:14–18]. They obtained a higher level of cellulose production in Acetobacter xylinum upon expression of mung bean sucrose synthase. This bacterium lacks sucrose synthase so is limited to only the UGPase branch of the pathway for making UDP-glucose (FIG. 3). Expression of sucrose synthase also led to a higher level of UDP-glucose and a lower level of UDP in the bacterium, as would be expected based on the pathway in FIG. 3.
Down-regulation of SuSy by antisense approach in carrot reduced the growth rate [Tang, et al. (1999), “Antisense repression of sucrose synthase in carrot (Daucus carota L.) affects growth rather than sucrose partitioning,” Plant-Molecular-Biology 41:465–479]. Levels of UDP-glucose and cellulose were reduced in the sink tissues in comparison to the wild type plants, again implying a role for SuSy in substrate production for cellulose synthesis. In work with the TUSC (Trait Utility System for Corn; see U.S. Pat. No. 5,962,764, incorporated herein by reference) SuSy mutant, knocking out the constitutive sucrose synthase led to a reduced cellulose concentration in the walls, as well as reduced amount of total cell wall (Example 8).
Formation of UDP-glucose from sucrose requires half as much energy as if it were to be made from hexose sugars (FIG. 3). Not even accounting for the channeling effect, as suggested by Amor et al. [1995, supra], involvement of sucrose synthase in providing substrate to cellulose synthase would lead to improved productivity, particularly under stressful conditions, as the energy conserved by this pathway could be used for other cellular processes. Over expression of sucrose synthase under the control of a stalk-preferred promoter in plants could lead to a greater synthesis of cellulose, thereby strengthening the stalk. Therefore, there is a need in the art for sucrose synthases that can be over-expressed under these conditions.
Sucrose phosphate synthase may participate in UDP-glucose metabolism, but its role appears to be more to dissipate energy in the sink tissues than to economize the use of sugars (FIG. 3). For example, assuming that all the fructose-6-phosphate and UDP-glucose are derived from the SuSy pathway, at least one ATP is consumed to make sucrose from these two substrates only for the former to be cycled through SuSy again. On the other extreme, i.e., when all the UDP-glucose and fructose-6-phosphate are derived from hexose sugars, formation of sucrose by sucrose phosphate synthase utilizes 3 NTP per sucrose molecule produced, two to form UDP-glucose from a hexose sugar and 1 to phosphorylate fructose. In other words, involvement of sucrose phosphate synthase would consume an extra 1–3 NTP per molecule of sucrose to be incorporated into cellulose, which means a consumption of 3–5 net NTP for this process.
Four NTP would be needed per sucrose molecule for its complete conversion to UDP-glucose even if all the sucrose were first to be cleaved by invertases, and hexoses were the only sugars available. Even invertases dissipate (waste) the energy of the glycosidic bond which is otherwise used by sucrose synthase to form UDP-glucose from UDP. Sucrose phosphate synthase may, however, be important in mediating the formation of sucrose from excess hexoses for transport to other sinks, such as developing ear. This could be important after the deposition of cellulose into the walls of stalk tissue has slowed down.
Each hexose sugar molecule, upon complete breakdown by glycolysis, citric acid cycle, and oxidative phosphorylation, produces 36 ATP equivalents of energy. As discussed above, each hexose upon activation into UDP-glucose uses 1 ATP if carried through the SuSy pathway and 2 ATP if through the UGPase pathway (FIG. 3). The fraction of sugar utilized, assuming all other processes to be constant, in supporting this conversion is:
      p    +          2      ⁢      q        36where p is the proportion of substrates produced by the action of SuSy; q represents substrates produced from hexose sugars; and p+q=1. If all the UDP-glucose were to be derived from the SuSy-mediated pathway, then 2.8% of the sugar would be utilized in producing energy to support this reaction. If, on the other hand, hexose was the starting point for all the UDP-glucose produced, then 5.6% of the sugar would be utilized in generating energy for this series of reactions.
Routing of any proportion, n, of the sugars through the sucrose phosphate synthase pathway would reduce the efficiency further still as the NTP utilized for this cycling would be in addition to the ones used in making UDP-glucose from sucrose or hexose. The following expression provides an estimate of the reduction in efficiency:
            (              p        +                  2          ⁢          q                    )        +          n      ⁡              (                  p          +                      3            ⁢            q                          )              36If 50% of the sugar is cycled through the sucrose phosphate synthase pathway and the substrates for this enzyme are derived in equal proportion (i.e., p=q=0.5) from the SuSy and UGPase pathways then, without including the energy needed for the sucrose phosphate synthase pathway to operate, this would translate into 4.2% of the sugar converted into cellulose being utilized for energy generation to support this process. If, however, the energy utilized by the sucrose phosphate synthase pathway, based on above assumptions, is taken into account, then this number increases to ˜7%, a full 70% extra energy than if no sugar were cycled through this pathway. That is equivalent to burning nearly 3 extra bushels of sugar for every 100 bushels converted into polysaccharides.
Thus, the production of cellulose through the sucrose synthase pathway is the most economical means available to plants. One of skill in the art would know of the involvement of sucrose synthase in cellulose formation in plants. However, the present invention teaches that this enzyme is important in supplying substrate for cellulose synthesis (Example 8).
As stalk composition contributes to numerous quality factors important in maize breeding, what is needed in the art are products and methods for manipulating cellulose concentration in the cell wall and thereby altering plant stalk quality to provide, for example, increased standability. It would be desirable to over-express sucrose synthase, preferably under the control of a stalk-preferred promoter, to improve stalk strength in maize.
Another attribute of importance is grain handling ability, i.e., reducing grain breakage during combining, transport, and movement into storage. Grain strength in cereals such as wheat and barley is mainly derived from the pericarp, which allows for a softer endosperm. It would be desirable to increase cellulose in the pericarp by over-expressing sucrose synthase under the control of a pericarp-specific promoter.
The present invention provides these and other advantages.